We have cloned gibberellin (GA) 3β-hydroxylase cDNA, (HvGA3ox), a homolog of the rice gene OsGA3ox from germinating barley seeds. HvGA3ox cDNA was expressed in Escherichia coli as a fusion protein capable of oxidizing GA₉ to GA₄ and GA₂₀ to GA₁. Deduced amino acid sequence analysis showed that HvGA3ox was identical to HvGA3ox2, which shares a high homology with the OsGA3ox2 isoform that plays an important role for induction of α-amylase in germinating rice seeds. Northern blot analysis showed that HvGA3ox2 gene was expressed only in germinating seeds, not in other organs. Typical feedback regulation was not observed in seedling treated with either biologically active GA (GA₃) or uniconazole, an inhibitor of GA biosynthesis. HvGA3ox2 mRNA was detected from 12 h after imbibition just before expression of α-amylase gene, Amy1. In situ hybridization of germinating seeds revealed that HvGA3ox2 mRNA was localized in the epithelium up to at least 3 days after imbibition. Our results suggest that GA biosynthesis in epithelium is important for expression of α-amylase in germinating barley seeds, and that HvGA3ox2 encodes the key enzyme of this event.